The role of the Shc phosphotyrosine interaction phosphotyrosine binding domain and tyrosine phosphorylation sites in polyoma middle T antigen-mediated cell transformation

The phosphotyrosine Interaction (PI)/phosphotyrosine binding (PTB) domain of She binds specific tyrosine-phosphorylated motifs found on activated growth factor receptors and proteins such as polyoma virus middle T antigen (MT), Phenylalanine 198 (Phe(198)) has been identified as a crucial residue involved in the interaction of the She PI/PTB with phosphopeptides. In MH3T3 cells expressing MT, p52 She carrying the F198V mutation is weakly phosphorylated and does not bind MT or Grb2. Overexpression of the PI/PTB domain alone as She amino acids 1-238 acted in a dominant interfering fashion blocking MT-induced transformation, However, expression of a slightly longer construct, She 1-260, which encompasses Tyr(239)/Tyr(240), a novel She tyrosine phosphorylation site, did not block transformation, This was found to be due to the ability of She 1-260 to become tyrosine-phosphorylated and bind Grb2. Furthermore, full-length She in which Tyr(239)/Tyr(240) had been mutated to phenylalanine did not become tyrosine-phosphorylated or bind Grb2 but did inhibit colony formation in soft agar, Conversely, p52 She carrying a mutation in the other tyrosine phosphorylation site, Tyr(317), became heavily tyrosine-phosphorylated, bound Grb2, and gave rise to colonies in soft agar.